CN110499252B

CN110499252B - Automated cell culture

Info

Publication number: CN110499252B
Application number: CN201910410428.6A
Authority: CN
Inventors: 荆杰; 肯尼斯·安德鲁·柯林斯; 袁方; 张中华; 劳伦斯·圣乔治; 杰弗里·詹姆斯·德里斯科尔; 谭维嘉; 蔡皓; 林东平; 程剑
Original assignee: Huacells Corp
Current assignee: Huacells Corp
Priority date: 2018-05-18
Filing date: 2019-05-16
Publication date: 2023-04-18
Anticipated expiration: 2039-05-16
Also published as: US20190352589A1; TW202003833A; EP3775156A1; CN110499252A; JP2023181365A; JP2021522826A; EP3775156A4; TWI765154B; WO2019219050A1

Abstract

An automated cell culture system comprising a cell culture reactor comprising a housing; a fluid circuit for a cell culture fluid, the fluid circuit being disposed inside the housing. The fluid circuit includes a culture vessel for culturing cells in a cell culture fluid, a reservoir for the cell culture fluid, the reservoir being in fluid connection with the culture vessel, and a pump configured to pump the cell culture fluid into the fluid circuit. The automated cell culture system includes one or more sensors disposed inside the housing, each sensor configured to detect one or more of (1) a cell culture fluid in the fluid circuit and (2) a parameter in an environment inside the housing; a computing device configured to automatically control operation of the cell culture reactor based on the one or more detected parameters.

Description

Automated cell culture

This application claims priority to U.S. patent provisional application serial No. 62/673,484, filed on 2018,

month

5, 18, and U.S. patent application serial No. 16/229,303, filed on 2018, month 12,21, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present application relates to the field of cell culture, in particular to automated cell culture.

Background

Cell culture can be used to increase cell number. For example, T cells can be extracted from an individual's blood and cultured in vitro to rapidly increase the number of T cells. By contacting with an immune activator such as an antigen or cytokine, the cultured T cells can be activated to exert a disease-resistant effect more effectively. The expanded and activated T cells can be injected into a human body to improve the immune response of the human body to a disease. Cell culture may also be used for other types of cells, such as stem cells or other types of cells; or for the production of cell-derived products, for example for the production of proteins, eukaryotic cell-derived products or other products.

Disclosure of Invention

An automated cell culture system is provided. In one aspect, the automated cell culture system includes a cell culture reactor and a housing; a fluid circuit for a cell culture fluid, the fluid circuit being disposed inside the housing. The fluid circuit includes a culture vessel for culturing cells in a cell culture fluid, a receptacle for the cell culture fluid, a fluid connection line between the receptacle and the culture vessel, and a pump configured to pump the cell culture fluid in the fluid circuit. The automated cell culture system includes one or more sensors disposed inside the housing, each sensor configured to detect one or more of (1) a parameter of a cell culture fluid in the fluid circuit, (2) an environment inside the housing; and a computing device configured to automatically control operation of the cell culture reactor based on the one or more detected parameters.

Implementations may include one or more of the following features.

The computing device is configured to control operation of the cell culture reactor based on comparing the detected individual single or multiple parameters and the stage of the cell culture. The computing device is configured to determine a stage of the cell culture from the one or more detected parameters.

The sensor includes a receptacle sensor configured to detect an amount of cell culture fluid in the receptacle. The receptacle sensor includes a weight sensor. The receptacle sensor includes a stress deformer.

An automated cell culture system includes a rotating rack that mounts cell culture vessels.

The automated cell culture system comprises a supply system comprising a supply tube, one end of which is connected to the fluid circuit and the other end of which is connectable to a source of cell culture fluid; and a supply pump connected to the supply pipe. The supply system comprises a temperature control system, which comprises a shell, an inner space of which is used for storing cell culture solution; and a temperature control module configured to cool or heat the inner space of the housing. The computing device regulates operation of the supply pump based on the amount of cell culture fluid in the receptacle. When the amount of cell culture fluid in the receptacle is less than a threshold amount, the computing device controls operation of the supply pump. The computing device controls the operation of the supply pump according to a target amount of cell culture fluid in the vessel, the target amount being set based on the stage of the cell culture. The computing device controls the operation of the supply pump based on the pH of the cell culture fluid in the fluid circuit.

The sensor includes one or more pH sensors configured to detect a pH value of the cell broth in the fluid circuit. The pH sensor comprises a colorimetric pH sensor. The pH sensor comprises an ionic pH sensor.

An automated cell culture system includes a heater disposed inside a housing. The computing device is configured to control operation of the heater based on a temperature of an outer wall of the culture vessel.

The automated cell culture apparatus includes a valve disposed in the interior space of the housing, and the computing device is configured to control operation of the valve based on a concentration of a gas inside the housing.

The automated cell culture system includes a gas source connected to the interior space of the device housing; comprising a gas flow control device connected to a gas source. The computing device is configured to control operation of the airflow control device based on the one or more detected parameters. The gas flow control apparatus includes a mass flow controller. The gas flow control device comprises a metering valve. The computing device controls operation of the gas flow control device based on a comparison between (i) a deviation between the gas concentration inside the housing and a threshold concentration and (ii) a target deviation. One or more sensors (gas sensors) detect the concentration of the gas inside the housing. The computing device is configured to control operation of the gas flow control device based on the detected gas concentration. The computing device is configured to control operation of the gas flow control device based on a pH of a cell culture fluid in the fluid circuit. The computing device is configured to control operation of the gas flow control device based on the dissolved oxygen amount of the cell culture fluid in the fluid circuit.

The one or more sensors include a dissolved oxygen sensor that detects an amount of dissolved oxygen in the cell culture fluid in the fluid circuit.

The one or more sensors include a glucose sensor that detects a glucose content of the cell culture fluid in the fluid circuit.

The one or more sensors include a lactate sensor that detects lactate content in the fluid circuit.

The computing device controls operation of the cell culture reactor based on a stage of cell culture within the culture vessel. The computing device determines a stage of the cell culture from one or more of (i) the one or more detected parameters and (ii) a history of the one or more detected parameters. The computing device controls operation of the pump according to the stage of the cell culture. The automated cell culture system includes a supply system including a supply line, a first end of the supply line connected to the fluid circuit, and a second end of the supply line connected to a source of cell culture fluid; and a supply pump coupled to the supply line; and a computing device therein that controls the operation of the supply pump according to the stage of the cell culture.

The culture vessel comprises a hollow fiber cartridge.

The automatic cell culture system comprises a waste liquid pipeline, wherein one end of the waste liquid pipeline is connected to the fluid loop, and the other end of the waste liquid pipeline is connected to the waste liquid container; and a waste pump coupled to the waste line.

The automated cell culture system includes a user interface, wherein the computing device is configured to cause an output on the user interface indicative of the one or more detected parameters. The user interface includes a graphical user interface. The user interface comprises a touch-sensitive user interface.

The computing system may output information to the remote computing device that displays the one or more detected parameters.

The computing device generates an alert output based on the one or more detected parameters.

The automated cell culture system includes a data storage device for storing information indicative of one or more detected parameters.

The computing device transmits an information indication of the one or more detected parameters to the data storage terminal over the network connection.

The present application also provides a method of culturing cells. In one aspect, the method of culturing cells comprises culturing cells in a cell culture reactor, including a cell culture fluid flowing in a fluid circuit internal to the cell culture reactor, including pumping the cell culture fluid in a receptacle to a cell culture vessel by a pump to culture the cells. The method includes detecting one or more parameters by one or more sensors inside the cell culture reactor (1) a parameter of one or more cell culture fluids in the fluid circuit, and (2) an environment inside the cell culture reactor; and automatically controlling, by the computing device, operation of the cell culture reactor based on the one or more detected parameters.

Implementations may include one or more of the following features.

Controlling operation of the cell culture reactor comprises comparing each detected parameter to a respective threshold value; and controlling the operation of the cell culture reactor based on the comparison result. The threshold value of the at least one detection parameter is based on the stage of the cell culture. The method determines the basis for the cell culture phase by including one or more of (i) the one or more detected parameters, (ii) a history of the one or more detected parameters.

The sensing parameter includes sensing an amount of cell culture fluid in the receptacle.

The method includes rotating the cell culture vessel.

The operational control of the cell culture reactor includes controlling the operation of the supply pump to pump the cell culture fluid from its source into the fluid circuit. The method includes controlling the temperature at the source of the cell culture fluid. The method includes heating a cell culture fluid added to a fluid circuit from a cell culture fluid source. The method includes controlling operation of the supply pump based on the amount of cell culture fluid in the receptacle. The method includes controlling operation of the supply pump based on the weight of the cell culture fluid in the vessel being less than a threshold amount. The method includes controlling operation of the supply pump based on a target amount of cell culture fluid in the vessel, the target amount being associated with a stage of the cell culture. The method includes controlling operation of the supply pump based on the pH of the cell culture fluid in the fluid circuit.

The method includes detecting the pH of the cell culture fluid in the fluid circuit.

The controlling operation of the cell culture reactor includes controlling the heater according to the temperature of the cell culture reactor.

Controlling operation of the cell culture reactor includes controlling operation of a gas flow control device connected to a source of gas. The method comprises detecting a gas concentration inside the cell culture reactor; and controlling the operation of the gas flow control device in accordance with the detected gas concentration. The method includes controlling operation of the gas flow control device based on a comparison of (i) a deviation of the gas concentration inside the cell culture reactor from a threshold concentration and (ii) a target deviation. The method includes controlling operation of the gas flow control device based on the pH of the cell culture fluid in the fluid circuit.

The method includes controlling operation of a valve in the cell culture reactor based on the detected gas concentration inside the cell culture reactor.

The method includes controlling operation of the cell culture reactor based on the stage of cell culture in the culture vessel. The method includes determining a stage of the cell culture based on the one or more detected parameters. The method includes controlling the manner in which the cell culture fluid is pumped in the fluid circuit according to the stage of the cell culture. The method includes controlling the operation of the supply pump to pump cell culture fluid from a source thereof into the fluid circuit according to the stage of the cell culture.

The method includes triggering an alert output based on one or more detected parameters.

The method includes outputting information of the detected one or more parameters on a user interface. The information output on the user interface includes an output of graphical information. The method includes receiving an information input through a user interface; and controlling operation of the cell culture reactor based on the received input.

The method includes conveying information of the one or more detected parameters to a data storage terminal.

The method includes transmitting information of the one or more detected parameters to a data storage device over a network connection.

The method includes network connecting, by a remote computing device, the computing device to control operation of the computing device.

Automated cell culture systems may have one or more of the following advantages. The cells can be cultured automatically under computer control, the process does not involve labor-intensive manual operations, and the risk of mishandling and contamination is low. In an automatic cell culture system, the closed cell culture process can reduce or minimize human factor interference, and facilitates large-scale, low-cost and clinical-level manufacturing. The automatic cell culture system adopts a sealed cell culture fluid loop, can be used as a desktop computer with a compact structure, occupies small space and has high cost performance.

Drawings

FIGS. 1 and 2 are schematic diagrams of a cell culture system.

Fig. 3 is a schematic view of a hollow fiber cartridge.

Fig. 4 is a schematic diagram of a fluid circuit.

Fig. 5 and 6 are flowcharts.

Fig. 7 is a schematic diagram of a pH sensor.

Fig. 8 is a flowchart.

FIG. 9 is a schematic diagram of a temperature control subsystem.

Fig. 10 is a schematic diagram of a gas control subsystem.

Fig. 11 is a flowchart.

FIG. 12 is a schematic of a cell culture system.

Fig. 13A-13C are schematic views of a rotating gantry.

Fig. 14A-14C are schematic views of a rotating gantry.

Fig. 15A-15C are schematic views of a rotating gantry.

FIG. 16 is a schematic diagram of a temperature control system.

Fig. 17A and 17B are schematic diagrams of a temperature control system.

FIG. 18 is an overview of a user interface.

Fig. 19 is a screen shot.

FIGS. 20A-20C are screen shots.

Fig. 21 and 22 are screen shots.

FIGS. 23A and 23B are graphs of cell numbers.

Fig. 24A and 24B are graphs of glucose and lactate concentrations.

FIGS. 25A-25C are graphs of cell expansion curves.

FIGS. 26A-26E are graphs of cell expansion curves.

FIGS. 27A-27E are graphs of cell expansion curves.

Detailed Description

We describe an automated cell culture system that is computer controlled and allows for automated cell culture, transfection and expansion, suitable for use with commonly suspended or adherent cells, such as T cells, stem cells, or other types of cells. The cell culture environment in an automated cell culture system can be dynamically and automatically adjusted based on real-time monitoring of sensors in the system to maintain steady-state levels of cell culture fluid, e.g., maintaining parameters such as culture fluid volume, fluid pressure, flow rate, pH, dissolved oxygen, glucose concentration, lactate concentration, or other parameters within a predetermined combination. Maintaining a steady state level of the cell culture solution helps to reduce the physicochemical stress on the cultured cells, thereby improving the culture efficiency and cell viability. The product obtained from an automated cell culture system may include the cells themselves or byproducts of the cell culture, such as proteins, viruses, antibodies, etc.

The automatic cell culture system can be used for culturing various cells, including cell culture in atmospheric environment; culturing cells in a hypoxic environment; culturing in serum containing human or animal origin; serum-free cell culture; culturing T lymphocyte, red blood cell, induced pluripotent stem cell, natural killer cell, cell line and the like; and other processes. The activation and/or expansion of living cells using the automated cell culture system can modulate the cell growth rate to increase the harvest rate.

Referring to fig. 1, an automated cell culture system 100 is an integrated apparatus for automatically culturing cells under computer control. The base 102 of the automated cell culture system 100 contains a computing device 101 (e.g., a computing device having one or more microprocessors coupled to memory) that controls the operation of the automated cell culture system 100. The chassis cover 103 may close and mechanically lock the base 102, for example, to help prevent accidental or unauthorized access to the computing device 101.

The reactor space 104 of the automated cell culture system 100 comprises an inner space constructed by the housing 105, in which the cell culture is performed. The housing may be sealed. For example, the housing may be mounted on the chassis cover 103 by a silicone gasket. The sealed housing may help prevent gas leakage, for example, enabling effective control of gas concentration and effective gas usage inside the housing 105, and efficient temperature control. The reactor space 104 houses a cell culture vessel in which cells, such as T cells, can be efficiently cultured in a cell culture fluid in an environment monitored and controlled by a computing device. The reactor space 104 may house one or more sensors coupled to a computing device of the automated cell culture system 100. The sensor may detect a parameter of the culture environment, such as temperature, pH of the cell culture fluid, concentration of a gas, such as oxygen or carbon dioxide, in the atmosphere of the reactor space 104, concentration or partial pressure of a gas in the cell culture fluid, such as dissolved oxygen, concentration of a sugar, such as glucose, lactate, or other parameter of the cell culture environment. The reactor space 104 may house one or more components that may operate under the automated control of computing equipment of the automated cell culture system 100, such as heating components, gas flow controllers, pumps, or other components of the automated cell culture system 100. For example, a computing device of the automated cell culture system 100 may automatically control the operation of one or more components in the reactor section 104 in a closed-loop feedback system based on one or more detected culture environment parameters.

Automatic monitoring and control of culture environment parameters helps to achieve efficient cell culture. For example, components of an automated cell culture system can be controlled in real time based on changes in cell culture environment parameters without waiting for a user to manually operate the system or manually enter control instructions. The real-time response to the real-time detection ensures that the target parameters, such as the set target weight of the cell culture fluid, the set target concentration of the gas, the set target concentration of other culture additives in the system, such as growth factors, target temperature, target pH, or other parameters, are continuously stable. The target parameters can be kept constantly stable at the set values throughout the cell culture process, which helps to improve the culture efficiency.

Automatic monitoring and control of culture environment parameters also helps to achieve efficient use of resources (e.g., growth factors), thereby reducing material costs associated with cell culture. For example, based on real-time monitoring of culture environment parameters, the stage at which the cell culture process is occurring can be determined. The target values for certain parameters may vary from stage to stage of the cell culture process. These parameters can be monitored and adjusted in real time to dynamically respond to the determined stage of the cell culture process.

In the example of fig. 1, a computing device 101 installed in an automated cell culture system base 102 controls the operation of the automated cell culture device 100, e.g., receives signals from one or more sensors in a reactor and controls the operation of one or more components installed in a reactor space 104. In some examples, automated cell culture apparatus 100 may be connected by a wired or wireless connection to a remote computing device, such as a laptop or desktop computer, a server, or a mobile computing device, which may monitor and/or control the operation of automated cell culture apparatus 100. In some examples, a microprocessor-based controller installed in the base 102 of the automated cell culture system, or a controller connected to the automated cell culture system, may monitor and/or control the operation of the automated cell culture system. As used herein, the terms "computer controlled" and "controlled by a computing device of an automated cell culture system" and the like refer to a controller installed in the automated cell culture system 100 or by a remote computing device or microprocessor-based.

A display 106, such as a Liquid Crystal Display (LCD), may be mounted or integrated on the automated cell culture system 100, such as on the housing of the cassette lid 103 or on the housing of the reactor space 104. Display 106 may be controlled by a computing system to display parameter information of the cell culture environment, such as real-time readings from one or more sensors. The display 106 may be controlled by the computing system to display a status alert, e.g., indicating that a certain parameter has exceeded or fallen below a threshold. In some examples, the information may be displayed on a remote display, such as on a remote computing device, which may be a laptop computer or a mobile computing device, the display being connectable to the computing device of the automated cell culture system 100 by wired or wireless means. In some examples, the status information may be presented in other manners, such as by a visual indicator (e.g., one or more lights that blink or illuminate in a specified pattern to convey the status information) or an audible indicator (e.g., using an alarm or voice to convey the status information).

In some examples, the display 106 can be an interactive display, such as a touch-sensitive display, capable of receiving input from a user and delivering a signal indicative of the input information to the computing system. For example, the display 106 may receive user-provided information or instructions, such as: instructions to set a threshold level for a parameter of a cell culture environment; operating instructions for one or more components in the reactor space 104; information identifying or describing cells to be cultured in the automated cell culture system 100; or other types of information or instructions. In some examples, the information or instructions may be received from a remote computing device, for example, a laptop computer, desktop computer, or mobile computing device connected to the computing device of automated cell culture system 100 by wired or wireless means. Such information may be stored in a centralized data store (e.g., a cloud-based data store) or in a distributed data storage system. This information may be analyzed to improve system performance, fine tune parameter threshold settings, or for other purposes. For example, using the collected information, artificial intelligence based cell culture algorithms can be developed or upgraded and applied to automated cell culture systems.

Referring to FIG. 2, cells are cultured in a culture vessel 200, such as a cartridge (e.g., a hollow fiber cartridge), in a reactor 104 inside an automated cell culture system 100. Culture container 200 is connected to receptacle 202, such as a bottle, via a fluid line (not shown), such as a silicone tubing. Pump 204, such as a peristaltic or pulsatile pump, delivers cell culture fluid from receptacle 202 through a first portion of tubing to the input of culture vessel 200, and from the output of culture vessel 200 through a second portion of tubing back to the receptacle of the fluidic circuit (e.g., fluidic circuit 400 shown in FIG. 4 below).

Fresh cell culture fluid is introduced into the fluid circuit through a supply line from a fresh culture fluid source 206. For example, a pump 208a (e.g., a peristaltic pump) and one or more valves 210 (e.g., pinch valves) are operated by computer control to pump fresh broth from its source 206 through the supply line into the fluid circuit. The cell culture fluid may be driven by a pump 208b (e.g., a peristaltic pump) to exit the fluid circuit along a waste line and output to a waste receptacle (not shown). The

pumps

208a, 208b may be operated automatically under the control of the computing system of the automated cell culture system. In some instances, the pumping of fresh cell culture fluid from its source 206 into the fluid circuit may occur simultaneously with the draining of cell culture fluid from the fluid circuit into the waste receptacle to avoid providing cell culture fluid in excess of the capacity of the fluid circuit. In some examples, fresh cell culture fluid is pumped into the fluidic circuit from its source 206 at a higher flow rate than the flow rate of fluid discharged from the fluidic circuit into the waste receptacle to increase the volume of cell culture fluid in the fluidic circuit. In some examples, cell culture fluid from the fresh culture fluid source 206 is injected into the fluidic circuit at a lower flow rate than the flow rate of the cell culture fluid discharged from the fluidic circuit to the waste destination to reduce the volume of cell culture fluid in the fluidic circuit.

One or more sensors installed in the reactor space 104 of the automated cell culture system 100 detect parameters of the culture environment in real time, producing signals indicative of the delivery to the local controller and/or computing devices of the automated cell culture system 100. The sensor may be in wired or wireless communication with a computing device of the automated cell culture system. The culture environment refers to the cell culture fluid and the internal atmosphere of the housing 104 of the automated cell culture system 100. These sensors may include temperature sensors, pH sensors, dissolved gas sensors, atmospheric gas sensors, glucose sensors, lactate sensors, fluid weight or volume sensors, or other types of sensors. The controller or computing device 100 may automatically control the operation of one or more components of the automated cell culture system (e.g., heaters, gas flow controllers, pumps, or other components) in response to the detected parameters without real-time user input. Real-time adjustment of parameters (e.g., via a closed-loop feedback system) can result in time-efficient cell culture. In some examples, the computing device 100 may determine the stage of the cell culture based on the detected parameters, or a history, or both, to further control the operation of one or more components of the automated cell culture system 100. When the perception parameter exceeds or falls below a threshold, or the cell culture phase changes, or for other reasons, the computing device 100 may output an alert (e.g., one or more of a visual alert and an audio alert) on a user interface (e.g., on the display 106). .

In some examples, one or more pH sensors 212,214 may be positioned in the reactor space 104 of the automated cell culture system 100 and positioned to detect the pH of the cell culture fluid in the fluid circuit. For example, pH sensors 212,214 may be positioned in the tubing between receptacle 202 and the input of culture vessel 200, or in the tubing between the output of culture vessel 200 and receptacle 202, or elsewhere in the fluid circuit, to detect the pH of the cell culture fluid. In the example of fig. 2, pH sensor 212 is an ionic pH sensor that detects the pH of the broth based on the photoluminescence quenching of the liquid, and pH sensor 214 is a colorimetric pH sensor that detects the pH of the fluid based on the Hue value of the fluid being measured. Other types of pH sensors may also be used. In some examples only a single pH sensor may be used.

The pH sensors 212,214 may communicate with the computing system of the automated cell culture system 100 by wired or wireless communication. For example, when the pH of the cell culture fluid is below a pH threshold (e.g., a user-set threshold, or the pH is below a set pH that causes cell culture efficiency to decrease, or the pH is below a set pH that is detrimental to the cultured cells), the computing system of the automated cell culture system 100 can automatically control the pump 208b to pump the cell culture fluid from the fluid circuit to the waste receptacle, and then can automatically control the pump 208a to pump fresh cell culture fluid from the fresh culture fluid source 206 to the fluid circuit; the carbon dioxide gas flow controller can also be automatically controlled to reduce the concentration of carbon dioxide in the gas inside the reactor part; other components of the automated cell culture system 100 may also be automatically controlled. When the pH value is below the pH threshold, the computing system may output an alert on a user interface (e.g., on display 106).

The automated cell culture system 100 includes a thermal control subsystem in the reactor space 104 that can monitor and control the temperature inside the reactor space 104 of the automated cell culture system 100. Temperature sensor 216 senses the temperature of the outer wall of culture vessel 200 and is indicative of the temperature of the cell culture fluid in the fluid circuit. One or more heating devices (e.g., a heater, a fan, or both) in heater 218 may be operated under the control of a computing system of an automated cell culture system to control the temperature of the exterior walls of culture vessel 200 according to a closed loop feedback system of the temperature detected by temperature sensor 216. When the temperature falls within the preset temperature range, the computing system may output an alert on a user interface (e.g., display 106). The thermal control subsystem is discussed in more detail in fig. 9.

The automated cell culture system 100 includes a pneumatic control subsystem in the reactor space 104 for monitoring and controlling the concentration of one or more gases (such as one or more of oxygen, carbon dioxide, or other types of gases) inside the reactor of the automated cell culture system. One or more gas sensors (such as an oxygen sensor, a carbon dioxide sensor, or a sensor for another type of gas) detect the gas concentration in the air inside the reactor. The dissolved oxygen sensor 222 can detect the concentration of oxygen dissolved in the cell culture solution. For example, a dissolved oxygen sensor can determine the amount of oxygen present in the cell culture fluid surrounding the sensor based on fluorescence quenching of light.

The gas sensor communicates with the computing system of the automated cell culture system 100 by wired or wireless means. The computing system controls one or more gas flow controllers to supply gas (e.g., carbon dioxide or nitrogen) into the interior space where the culture system reactor is located. The gas flow supply is automatically regulated depending on the concentration of the gas (e.g., carbon dioxide or oxygen) in the space where the reactor is located, such as by a closed loop feedback system. In an example, the computing system can also adjust the gas flow controller based on detecting a concentration of a dissolved gas (e.g., dissolved oxygen) in the cell culture fluid within the resulting fluidic circuit. When the concentration of any one of the monitored gases is outside of the predetermined concentration range, the computing system outputs an alarm signal on a user interface (e.g., display 106). For more details on the gas control subsystem, reference may be made to fig. 10.

The automated cell culture system 100 includes a glucose sensor 224, such as a sensor based on enzymatic and micro-electromechanical system (MEMS) electronics, for detecting the glucose level in the cell culture fluid. The automated cell culture system 100 includes a lactate sensor for detecting the amount of lactate in the cell culture fluid. In an example, a single sensor is configured to detect both glucose and lactate. Glucose sensors 224, lactate sensors, and other types of sensors are disposed within the reactor space 104 of the automated cell culture system 100. These sensors communicate by wire or wirelessly with the computing system of the automated cell culture system 100 to monitor glucose consumption and lactate production in the cell culture fluid as indicators of cell growth. When the concentration of glucose or lactate is outside of the preset concentration range, the computing system outputs an alarm signal at a user interface (e.g., display 106).

The automated cell culture system 100 includes a fluid level sensor (not shown) disposed within the reactor space 104 to detect the amount (e.g., weight or volume) of cell culture fluid in the receptacle 202. The liquid amount sensor may be a weight sensor (e.g., a strain gauge) for detecting the weight of the cell culture liquid in the vessel 202; the liquid weighing may also be a volume sensor (e.g., an optical sensor) for detecting the volume of cell culture liquid in the receptacle 202. In an example, other types of sensors may be used to detect the amount of cell culture fluid in the receptacle.

The liquid weighing sensor communicates with the computing system of the automated cell culture system 100 by wire or wirelessly. For example, when the cell culture fluid in the receptacle 202 (representing the total amount of cell culture fluid in the fluid circuit) is below a certain threshold, the computing system of the automated cell culture system 100 automatically controls the pump 208a to pump fresh cell culture fluid into the fluid circuit. The threshold is some fraction of the maximum capacity of receptacle 202, such as 80%,60%,50%,40% or other fraction of the maximum capacity. The threshold may be a volume, such as 100mL,200mL,300mL,400mL,500mL, or other volume. The threshold value setting may be changed based on the stage of cell culture as described below. When the amount of cell culture fluid in receptacle 202 is below a threshold, the computing system outputs an alarm signal at a user interface (e.g., display 106).

In one example, the automated cell culture system 100 includes a fluid level sensor 226 disposed inside or outside the reactor space 104 for detecting the amount (e.g., weight or volume) of fresh cell culture fluid in the receptacle 206. The liquid weighing sensor may be a weight sensor (e.g., strain gauge) for detecting the weight of the fresh cell culture solution; the liquid weighing can also be a volume sensor (e.g., an optical sensor) for detecting the volume of fresh cell culture fluid. In an example, other kinds of sensors may be used to detect the amount of fresh cell culture fluid. In an example, the automated cell culture system 100 may include a liquid weighing sensor disposed inside or outside the reactor space 104 for detecting the amount (e.g., weight or volume) of cell culture waste liquid discharged.

The fluid volume sensor 226 communicates with the computing system of the automated cell culture system 100 by wired or wireless means. For example, when the amount of fresh cell culture fluid in receptacle 206 falls below a threshold, the computing system of automated cell culture system 100 outputs an alarm signal at a user interface (e.g., display 106) to alert the user that it is necessary to add fresh cell culture fluid. The liquid amount sensor for detecting the amount of the cell culture waste liquid is also in communication with the calculation system by a wired or wireless manner. For example, when the amount of cell culture waste exceeds a threshold, the computing system of the automated cell culture system 100 outputs an alarm signal at a user interface (e.g., display 106) to alert the user that it is necessary to empty the waste receptacle or replace a new waste receptacle.

In an example, the computing system uses one or more detection parameters to determine the stage at which the cell culture is in, such as: a slow growth phase is initiated, a fast (e.g., exponential) growth phase, or a plateau phase where cell growth slows or stalls. These parameters are, for example: the level of glucose or lactate in the cell culture fluid, the dissolved oxygen concentration in the cell culture fluid, the rate of nutrient loss in the cell culture fluid in the fluid circuit, or other parameters that may be used to characterize the cell culture stage.

The computing system of the automated cell culture system 100 determines the stage of the cell culture process based on the one or more sensed parameters, or based on a change in the one or more sensed parameters over time. The computing system controls the operation of one or more components within the system depending on the stage of the cell culture process. For example, during the initial phase of cell culture, without user intervention, the computing system automatically controls pumps 212a and 212b to maintain the amount of liquid in the fluid circuit at a lower level, thereby maintaining the growth factors in the cell culture fluid at a higher concentration level. During the rapid cell growth phase, the computing system automatically controls pumps 212a and 212b to increase the amount of liquid in the fluidic circuit without user intervention. The computing system controls the addition of cell culture reagents (e.g., cell growth factors) based on the stage of the cell culture. The computing system controls the operation of heater 218 during a particular phase of cell culture to maintain a corresponding target temperature within cell culture vessel 200. The computing system controls one or more gas flow controllers or gas valves (or different combinations thereof) to supply carbon dioxide or nitrogen to the reactor space at a particular stage of the cell culture to maintain carbon dioxide or oxygen in the space at a corresponding target concentration level or to maintain the pH or dissolved oxygen value of the cell culture fluid at a corresponding target level. The computing system outputs an alarm signal on a user interface (e.g., display 106) that may indicate the stage of the cell culture, indicate that the cell culture has reached a plateau, prompt the user for manual intervention (e.g., adding reagents such as growth factors, harvesting cultured cells, or other action), prompt the user that the system is taking action by itself (e.g., adding reagents such as growth factors, replacing fresh medium, or other action), or for other reasons, etc.

Referring to fig. 3, a hollow fiber cartridge 300 is a cell culture container for an automated cell culture system. The hollow fiber cartridge 300 is packaged in a sterile, self-contained package (e.g., sterilized by gamma irradiation) and can be used as a disposable cartridge consumable.

The hollow fiber cartridge 300 includes a housing 302 defining an interior space. A plurality of tubes (i.e., capillaries 304) are disposed in the interior space of the housing 302, and the longitudinal axis of the capillaries 304 is substantially aligned with the longitudinal axis of the housing 302. The capillary 304 is synthesized from a material that supports cell growth (e.g., polymer: polysulfone). The material processing may create porosity, thereby forming a plurality of through-holes in the walls of the capillaries. The capillaries 304 are fixed in the housing 302 by plugs 306 at both ends of the housing 302, and an inner space (referred to as a tube inner space) of each capillary 304 is connected to an inlet 308 and an outlet 310 of the cartridge 300 so that the inside thereof can be in fluid communication. Cartridge 300 is connected to the fluid circuit through inlet 308 and outlet 310.

The space formed by the exterior of the capillary 304 and the interior of the housing 302 is referred to as the tube exterior space 314. Cell culture occurs in the tube outer space 314. The tube plenum 314 is in fluid communication with an inlet fitting 316 and an outlet fitting 318, for example, the

fittings

316 and 318 may be barb fittings that extend through the housing 302 of the cartridge 300.

Connectors

316 and 318 connect to tubing to provide cell culture reagents such as cells, serum, growth factors, immune stimulants (e.g., cytokines) and other high molecular weight substances. Because the walls of the capillary tube 304 are porous, nutrients can enter the tube exterior space 314 from the tube interior space to support cell culture, while metabolic waste can also enter the tube interior space from the tube exterior space 314 and exit the cartridge 300 by the flow of cell culture fluid in the circuit. The size of the tube wall through-holes is determined by the size of the molecules passing between the tube interior space and the tube exterior space 314. For example, the pore size may be selected to allow passage of molecules having a molecular weight between 10kDa and 0.2 μm (e.g., 10kDa, 20kDa, 50kDa, or 0.1 μm).

The space 314 outside the tubes where the cells are cultured has a capacity limitation. For example, the volume of the tube void 314 can be between about 10mL and 100mL (e.g., 10-70mL,30-70mL,50-70mL,30-50mL, or other volume ranges). The small volume of the tube outer space 314 allows for cell culture reagents such as growth factors, serum or other reagents to be at relatively high concentrations, thereby promoting efficient cell growth. In an example, the small volume of the tube outer space 314 also causes the cultured cells to be in close proximity to each other, and facilitating cell-to-cell communication can also improve the efficiency of the cell culture. In some instances, cell culture may also occur in the intraductal space, while cell culture reagents (e.g., high molecular weight nutrients) remain ipsilateral to the cells, e.g., when cell production does not need to be large, or to enhance cell-to-cell communication, or for other reasons.

The cartridge characteristics such as volume, material, pore size, etc. can be tailored based on the cells being cultured. For example, the characteristics of the cartridge can be selected to achieve a target flow rate, gas transport rate, nutrient and waste exchange rate, and the like, as well as other aspects associated with cell culture, to promote efficient expansion of living cells.

The computing system of the automated cell culture system 100 regulates the amount of cell culture fluid in the fluid circuit depending on the stage of the cell culture. For example, the use of a relatively small volume of culture fluid at the beginning of the cell culture process allows for a higher concentration of cell culture reagents, such as growth factors, within the space 314 outside the hollow fiber cartridge 300, which promotes cell growth. The amount of the cell culture solution can be increased at the later stage of cell culture.

Referring to FIG. 4, in a fluid circuit 400 of an automated cell culture system, a pump 204 pumps cell culture fluid from a vessel 202 into a culture vessel 200 and back into the vessel 202, thereby maintaining circulation of the cell culture fluid in the fluid circuit 400. The cell culture fluid receptacle 202 in the fluid circuit 400 may be a bottle or other container. For example, receptacle 202 may be 500mL in volume. The receptacle 202 is sealed and has a vent to allow it to equilibrate to atmospheric pressure. The vent comprises an air filter 402, such as a 0.2 μm filter.

Receptacle 202 is connected to cell culture container 200 by tubing 404 (e.g., silicone tubing, 1/4 inch outer diameter). The characteristics of the tubing, such as wall thickness, length, etc., are tailored to the cells being cultured. For example, the line characteristics can be selected to achieve a target flow rate, gas exchange rate, nutrient and waste exchange rate, and the like, as well as other aspects associated with cell culture, to promote efficient expansion of living cells.

Cell culture fluid is pumped from receptacle 202 to culture vessel 200 via a line of fluid circuit by pump 204 and returned from culture vessel 200 to receptacle 202. For example, a cell culture fluid may be circulated to the space inside the tubes of the hollow fiber cartridge 300 through a fluid circuit to deliver nutrients to the cultured cells while discharging metabolic waste out of the cartridge. The pump 204 is a peristaltic pump, such as a finger-type peristaltic pump, such that the same line may be used continuously for days or weeks, e.g., up to two weeks, one month, two months, three months, etc.

The cell culture fluid pumped out of receptacle 202 passes through gas exchange line 408 before reaching culture vessel 200. Gas exchange line 408 is made of a material that can meet the gas exchange metabolic parameters required to culture the cells. For example, gas exchange line 408 is a platinum cured silicone rubber. One or more sensors are disposed along line 404 on the conduit between receptacle 202 and culture vessel 200, such as a colorimetric pH sensor 214, or a sensor combination 410 comprising one or more types of pH sensors, dissolved oxygen sensors, glucose sensors, lactate sensors, and the like.

Cell broth is pumped from the fresh broth container 206 into the fluidic circuit 400 by a pump 208a (e.g., a peristaltic pump) via an input line 412. Cell culture fluid is pumped from the fluid circuit 400 by a pump 208b (e.g., a peristaltic pump) through an output line 418 to a waste reservoir 416. The process of pumping fresh culture fluid from the receptacle 206 into the fluid circuit 400 and pumping cell culture fluid from the fluid circuit 400 into the receptacle 416 is referred to as exchange of cell culture fluid. The three design of fluid circuit 400 and inlet and

outlet lines

412, 418 ensure that the exchange of cell culture fluid does not interfere with the cell culture in culture vessel 200. For example, actuation of valves 420, 422, and 424 (e.g., pinch valves) may allow for the exchange of cell culture fluids without substantially disturbing the cells. For example, valve 420 is open while valves 422 and 424 are closed during cell culture fluid exchange. In an example, a part of the culture vessel (e.g., the space in the tube of the hollow fiber cartridge 300) may be washed with the culture solution at a low speed when the cell culture solution is exchanged.

Other cell culture related substances, such as cells, serum, growth factors, and other high molecular weight substances (collectively referred to as reagents) may be supplied directly to culture vessel 200 via input line 426. For example, the input line 426 communicates with the outside of the tube of hollow fibers 300.

The fluid circuit 400 is a sealed fluid circuit. To maintain its seal, the connection into and out of the fluid circuit 400 is a one-way connection. For example, the connection to the inlet line 412 of the fresh broth receptacle 206 is a one-way valve, so that fluid can only flow into the fluid circuit 400; the connection to the outlet line 418 of the waste receptacle 416 is a one-way valve, so that fluid can only flow out of the fluid circuit 400; the connection of the input line 426 for the input of reagent is a one-way valve, so that the substance can only flow into the fluid circuit 400. To maintain the sealing of the fluid circuit 400, when the fresh broth receptacle 206, the waste liquid receptacle 416 and the reagent receptacle are replaced, these receptacles are thermally welded to the

lines

412, 418 and 426 by welding

points

434, 436 and 438, respectively. Thermal welding uses thermoplastic tubing, such as polyvinyl chloride (PVC) tubing, which is connected without exposure to the atmosphere.

In an example, the exchange of cell culture fluid can be automated under computer control based on detected cell culture fluid parameters (e.g., pH, dissolved oxygen, amount of cell culture fluid in receptacle 202, and other parameters). For example, if the pH of the cell culture fluid or the amount of cell culture fluid in the receptacle is below a respective threshold, the system may control the pump 208a to pump additional fresh cell culture fluid from the receptacle 206 into the fluid circuit 400. In an example, the exchange of cell culture fluid may be automated under computer control based on the determined cell culture stage. For example, the threshold amount of cell culture fluid in receptacle 202 may depend on the stage at which the cells are being cultured. If the amount of cell culture fluid in vessel 202 during a particular phase of cell culture is below the threshold for that phase, the system may control pump 208a to pump additional fresh cell culture fluid into fluid circuit 400.

The rate at which the cell culture fluid circulates in the fluid circuit 400 may be controlled by the computing system, such as by controlling the operation of the pump 204. For example, the rate of circulation of the cell culture fluid in fluid circuit 400 may be dependent on the stage of the cell culture, e.g., a low flow rate at an early stage so that the cells are not disturbed, and a high flow rate at a later stage (fast growth stage) so that nutrients are efficiently supplied to the cultured cells while metabolic waste is also removed from cell culture vessel 200. The pump 204 may be automatically controlled by the computing system without user intervention, depending on the stage (determined by the computer) the cell culture is in.

Referring to FIG. 5, an example of the trigger change during the change of the culture medium is shown. Before starting the change, the weight of the fresh broth holder is checked to confirm that a sufficient amount of fresh broth supports the completion of the change. If there is not enough fresh broth, an alarm is triggered, such as may be displayed on a user interface. While checking the available capacity of the waste receptacle to confirm that there is sufficient space to accommodate the waste liquid discharged during the liquid change. For example, the accumulated waste discharge is compared to the maximum capacity of the waste receptacle and an alarm is triggered if the waste receptacle does not have sufficient space.

Referring to fig. 4, pump 204 continues to maintain circulation of the cell culture fluid within the fluid circuit during the fluid change.

Valves

420, 422, and 424 are configured to open, close, and close, respectively. The output pump 208b is activated until the volume of the cell culture fluid (e.g., weight or volume) in the vessel 202 is reduced to or exceeds the volume of the fluid change. The input pump 208a is then activated until the volume of cell culture fluid in the receptacle 202 increases to or exceeds the volume of the exchange fluid. The total amount of change will be recorded as the cumulative effluent discharge.

In an example, the fluid change control can occur periodically. Every time the liquid changing operation is timed by a logic timer, and the alarm is triggered when the liquid changing operation is not completed within the set time.

Referring to fig. 6, an example of a culture vessel (containing a hollow fiber cartridge) wash, such as to elute impurities or contaminants from the hollow fiber cartridge (such as introduced during manufacture of the hollow fiber cartridge). The washing process is automated by computer control and the user can also specify the flow rate and duration (input from the user interface). The user may also input instructions indicating one or more fluids to be used in the cleaning process.

The cleaning process is initiated upon receipt of a user command (600). At the beginning of the cleaning process, the weight of an empty receptacle (e.g., receptacle 202 in FIG. 2) may be cleared (602). For example, the user receives a prompt to select whether to zero the starting weight of the empty receptacle. Once cleared, the empty container weight parameter (e.g., a default value may be stored in a memory space or database table, etc.) is set to the current actual measured weight value (604). If there is no clear operation, the empty vessel weight parameter is still using the default value (606). In an example, a user may decide a default value for a weight parameter of an empty container; the user may also determine the type of empty container used, and different container types will correspond to their respective default weight parameters.

After the weight of the empty receptacle is set, the cleaning process starts (600). Referring to FIG. 4, the process begins (608) with filling the culture container 200 (e.g., a hollow fiber cartridge), pumping fresh cell culture fluid from the reservoir 206 into the reservoir 202 with the pump 208a, and circulating cell culture fluid from the reservoir 202 to the culture container 200 with the pump 204. After culture vessel 200 is filled with cell culture fluid, pump 204 continues to maintain circulation of the cell culture fluid within fluid circuit 400. During the initial phase of the cleaning,

valves

420, 422 and 424 are all open.

When the cell culture fluid in the receptacle 202 reaches the target amount, the pump 208a stops pumping the cell culture fluid from the receptacle 206.

Valves

420, 422 and 424 are configured to close, close and open, respectively, to fill the tube exterior space 314 of culture vessel 200 (e.g., a polysulfone culture tube). Over a period of time, such as 5 minutes,

valves

420, 422 and 424 are set to open, close and close, respectively, to facilitate environmental equilibration within the cartridge.

The first equilibration of culture vessel 200 is maintained for a predetermined period of time, and a cell culture broth change is initiated. At this point, pump 208b is activated to transfer cell culture fluid from receptacle 202 to waste receptacle 416. Then, the second equilibration of the culture vessel 200 is performed, which is twice as long as the first equilibration.

The volume of cell culture fluid changed (e.g., weight or volume) and the remaining volume of waste receptacle 416 may be monitored during the equilibration process to help ensure that receptacle 202 is not affected by an overflow of waste receptacle 416 and to ensure that receptacle 202 itself is not overfilled.

Referring to fig. 7, an example colorimetric pH sensor 700 detects the pH of a fluid, such as a cell culture fluid in a fluid circuit of an automated cell culture system. The colorimetric pH sensor includes a sensor holder 702 for holding a colorimetric sensor assembly 704 and an optical channel 706. The clamp 708 clamps the colorimetric sensor 700 to a tubing line (e.g., the tubing line 404 of the fluidic circuit 400, see fig. 4) such that the tubing line is disposed between the two portions of the optical channel 706. The optical channel 706 and colorimetric sensor assembly 704 detect the Hue value of the cell culture fluid (containing a pH sensitive stain such as phenol red) in the fluid circuit line. This value is returned to the computing system of the automated cell culture system. The computing system compares the measured Hue value of the fluid to a reference (e.g., a four-way transfer function, a deterministic equation, or a data look-up table of sufficient resolution) to determine the pH of the fluid.

Referring to fig. 8, the process of changing the cell culture fluid in an automated cell culture system may be based on the pH of the cell culture fluid in the fluid circuit. For example, the pH of the cell culture solution decreases as the cell culture progresses. In order to maintain the pH of the cell culture fluid within a predetermined range or above a predetermined threshold, the fluid change may be based on the pH of the cell culture fluid. In an example process of changing the cell culture fluid, a colorimetric pH sensor (e.g., sensor 700 in fig. 7) detects the pH of the cell culture fluid (800), which can also be detected by an ionic sensor (802), and other types of pH sensors can be used to detect the pH. In an example, the pH of the cell culture fluid can be detected by a single pH sensor. One or more detected pH values are compared to a pH threshold (804), which is either default to the system or set by the user himself. The measured pH value compared to the threshold value may be determined by the pH values detected by the plurality of sensors, such as using an average of them. If the detected pH of the cell culture fluid is less than (or equal to or less than) the pH threshold (806), a fluid change of the culture fluid is initiated (see FIG. 5, supra). If the detected pH of the cell culture fluid is greater than (or equal to or greater than) the pH threshold (806), a fluid change will not be initiated while the one or more pH sensors and the local or remote computing system continue to detect the pH of the cell culture fluid. In the example, pH monitoring is a continuous real-time process. The pH monitoring may also be performed at regular intervals, such as every 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, or other time period.

Referring to fig. 9, there is an example of a thermal control subsystem 900 that monitors and controls the temperature within the reactor space of an automated cell culture system. The culture container (e.g., hollow fiber cartridge) is placed inside a case cover 902 (e.g., double metal case cover, aluminum). The cover 902 here is also the cover 105 of the reactor space of the automated cell culture system (see fig. 1). The cover is insulated to help maintain the ambient temperature inside it stable at a target temperature suitable for cell culture. The lid is partially or completely impermeable to one or more gases (e.g., carbon dioxide, oxygen, or other gases associated with cell culture) to help maintain the culture fluid within the cell culture vessel at a target dissolved gas concentration. The cover is hinged to the base for easy opening and convenient operation of the cell culture container.

Heating control subsystem 900 contains

temperature sensors

904 and 906, which are in intimate contact with the culture vessel. Here

temperature sensors

904 and 906 may be part of temperature sensor 216 (see fig. 2). The

temperature sensors

904 and 906 are fixed to the outer wall of the culture vessel 200 by means of a bracket device such as a molded silicone pad.

Temperature sensors

904 and 906 can directly sense the temperature of the culture vessel or provide a signal output to determine the actual ambient temperature. For simplicity, it is assumed that the temperature sensor can directly output the temperature measurement in both cases. For example, the temperature sensor may be a thermometer, thermistor, thermocouple, semiconductor-based sensor, or other type of temperature sensor.

In an example, a single temperature sensor may be used, or more than two temperature sensors may be used. The use of multiple temperature sensors can be switched to a redundant backup sensor in the event of a failure of one of the temperature sensors. In an example, a particular temperature sensor (e.g., sensor 904) is identified as the master temperature sensor from which the computing system of the automated cell culture system takes temperature readings. If the computing system finds that the primary temperature sensor is malfunctioning, the computing system switches to another temperature sensor (e.g., sensor 906) to take a temperature reading. In an example, the computing system may output an alarm signal (e.g., an audible alarm, a text alarm, or a graphical alarm) at a user interface of the local or remote computing system to alert the user of the sensor failure, as well as an alarm light or other form of alarm. In an example, the computing system may also output an alarm signal for other reasons, such as inconsistent readings from the two temperature sensors: the temperature readings differ by more than a percentage threshold (e.g., 2%,5%, or 10%, etc.).

Thermal control subsystem 900 includes a temperature controller 908 (e.g., a PID differential controller) that receives signals from

temperature sensors

904 and 906 and controls the operation of one or more heaters 910 and fans 912 based on the signals, e.g., in a closed loop feedback system to maintain the temperature of the culture vessel. For example, temperature controller 908 may control the temperature of the culture vessel to a preset temperature level (default or entered by a user through a local or remote user interface of the automated cell culture system). In an example, the temperature controller 908 may control the heater 910 by controlling a pulse width modulator to provide a safer heat output when the measured temperature and the preset temperature are not consistent. Fan 912 is configured to maintain air flow over the heater 910 and above the heating unit, as well as within the interior of the housing cover 902. In an example, the fan 912 may operate independently of the heater, which may provide a cooling function, maintain gas mixing within the lid 902, or maintain gas flow delivery to gas sensors, such as the

gas concentration sensors

156 and 162 and the gas temperature sensor 914. In the example, heater 910 includes an emergency shut-off sensor 918 that shuts off heater 910 when a failure of heater 910 is detected, thereby helping to prevent ignition of the components of the automated cell culture system and also helping to reduce the risk of burning a user.

Temperature controller 908 may be a separate component of the automated cell culture system, as shown in FIG. 9, or may be a module of computing system 916. In an example, depending on the type of

temperature sensors

904 and 906, temperature controller 908 may receive and buffer signals from

temperature sensors

904 and 906. When temperature controller 908 is a separate component, it may send temperature signals from

temperature sensors

904 and 906 to computing system 916. Computing system 916 may display the temperature-related information at a local or remote user interface output of the automated cell culture system. Computing system 916 may also process the received signals to determine whether the detected temperature has reached an alarm state, such as: a detected temperature above an upper temperature threshold, a detected temperature below a lower temperature threshold, and other alarm conditions. When an alarm condition occurs, the computing system may output an alarm signal (e.g., an audible alarm, a text alarm, a graphical alarm, a warning light, etc.) at a local or remote user interface of the automated system culture system to alert the user.

In the illustrated embodiment, the heating control subsystem 900 includes a temperature sensor 914 mounted near the fan 912 (e.g., at the inlet of the fan 912) to monitor the temperature of the circulating air within the cabinet cover 902. The signal from the temperature sensor 914 is transmitted to the temperature controller 908 (as exemplified in fig. 9) or directly to the computing system 916 of the automated cell culture system. The computing system controls the operation of heater 910 and fan 912 based on the temperature detected by temperature sensor 914. For example, when the temperature detected by temperature sensor 914 exceeds a first temperature threshold (e.g., 45 ℃, 46 ℃,48 ℃,50 ℃ or other temperature), computing system 916 sends a signal to heater 910 to stop heating to avoid overheating the interior environment of lid 902; when the temperature detected by temperature sensor 914 is below a second temperature threshold (e.g., 40 deg.C, 42 deg.C, 43 deg.C, 45 deg.C, or other temperature), computing system 916 sends a signal to heater 910 to resume heating. In an example, the first and second temperature thresholds may be the same.

Referring to fig. 10, there is an example of a gas control subsystem 150 monitoring and controlling the concentration of one or more gases in the air inside the automated cell culture system reactor space 104 (e.g., inside the chamber lid 902). For example, gas control subsystem 150 monitors and controls the concentration of carbon dioxide so that the cell culture fluid possesses a buffering function. In an example, the partial pressure of carbon dioxide can be controlled between about 0% and 6% (e.g., between 0.04% and 5%). The gas control subsystem 150 monitors and controls the oxygen concentration to regulate the oxidative stress of the cell culture to affect the growth and function of the cultured cells. In an example, the partial pressure of oxygen can be controlled to be between about 0% and 25% (e.g., between 0% and 21%) by nitrogen substitution. The gas control subsystem 150 may also detect and control the concentration of other gases besides carbon dioxide and oxygen.

Carbon dioxide sensor 156 detects the concentration of carbon dioxide inside cover 902. The computing system 916 of the automated cell culture system, or a remote computing system, receives the signal from the carbon dioxide sensor 156 and controls the operation of the carbon dioxide mass flow controller 158 in response to the signal to maintain the concentration of carbon dioxide within a predetermined range. For example, when the carbon dioxide concentration falls below a threshold level, computing system 916 may control mass flow controller 158 to introduce a carbon dioxide gas stream from carbon dioxide gas source 160.

The oxygen sensor 162 detects the oxygen concentration inside the tank cover 902. The computing system 916 of the automated cell culture system, or a remote computing system, receives the signal from the oxygen sensor 162 and controls the operation of the nitrogen mass flow controller 164 in response to the signal to maintain the oxygen concentration within a predetermined range. By increasing the nitrogen concentration inside the tank cap 902, the oxygen concentration therein may be reduced. For example, when the oxygen concentration rises above a set threshold, computing system 916 may control mass flow controller 164 to direct a flow of nitrogen from nitrogen source 166. In some instances, when ambient air cannot compensate for gas consumption, such as when excessive oxygen consumption is caused by a large amount of biomass during cell culture, a target oxygen concentration may be achieved by providing oxygen-enriched air into the tank cover 902. In an example, a differential controller (PID controller) may be used to control the concentration of oxygen.

Carbon dioxide, nitrogen or other gases may be introduced into the interior of the housing cover 902 through the port 168. One or

more purge valves

170, 172 may allow air to be directed into or out of the interior of tank cover 902, for example, when carbon dioxide or oxygen concentrations deviate significantly from preset thresholds. In some examples, the computing system may cause an alarm output (such as an audible alarm, a text or graphic alarm, a warning light, or other type of alarm on a user interface of the automated cell culture system or on a user interface of a remote computer device) to alert the user that the gas concentration deviates from a preset range.

FIG. 11 shows that in some examples, carbon dioxide (CO) is compared to a target concentration in the tank cap 902 ₂ ) Nitrogen (N) ₂ ) Or other gases may be supplied in excess. The calculation system can calculate the deviation Delta O between the measured oxygen concentration and the target oxygen concentration in the cell culture gas environment ₂ . The computing system may also calculate the deviation Δ CO of the measured carbon dioxide concentration from the target carbon dioxide concentration in the cell culture gas environment ₂ . If (1) the oxygen level is above the target oxygen level or the carbon dioxide level is below the target carbon dioxide level; (2) Deviation Δ O ₂ Exceeding threshold deviation (Dev _ O) or deviation Δ CO of oxygen level ₂ Above a threshold deviation (Dev _ C) of carbon dioxide level, the relay closes to open the vent valve 172. If the carbon dioxide or oxygen deviation still exceeds the threshold deviation, the second relay closes to open the vent valve 170 and activate the fan. The second relay may be opened and closed multiple times to open the exhaust valve 170 and activate the fan until both deviations do not exceed the respective threshold deviations.

In some examples, culture vessel 200 may be mounted on a rotating rack. The rotating holder enables the culture vessel 200 to rotate about one or more axes during the culture cycle. Rotational changes in the orientation of culture container 200 during the course of a culture cycle can promote redistribution of cells and prevent formation of cell clumps from adversely affecting the growth and health of the cells.

As shown in FIG. 12, a rotating rack 350 may be loaded into the automated cell culture system 100 in place of the static base of the culture vessel 200.

As shown in FIGS. 13A-13C, the exemplary rotating bracket 450 includes a housing 452 having a base 454 on which the culture vessel 200 can be mounted. A mechanical rotation mechanism 456 is fitted within the housing 452. In some examples, the rotation device may include a motor 455 linked to a gear set 457, arm linkage or other means for transmitting power from the motor to the culture container 200.

As shown in FIGS. 14A-14C, culture container 200 is secured to base 454 of rotating bracket 450 by base 458. Base 458 may contain temperature sensor 216 for sensing the temperature of culture container 200.

The rotating bracket 450 enables the culture vessel 200 to rotate end-to-end about an axis through the culture vessel 200. The axis may pass through the center of the culture container 200 or may be offset from the center of the culture container 200. In FIGS. 14A to 14C, a rotation of 90 ° is shown, and the culture vessel 200 can be rotated from the horizontal direction (FIG. 14A) to the vertical direction (FIG. 14C). Continued rotation may return the culture container 200 to the horizontal orientation but head-to-tail interchanged, e.g., in the initial position, the first end 460a of the culture container 200 is positioned on the left side of the rotating bracket 450 and the other end 460b of the culture container 200 is positioned on the right side of the rotating bracket 450; in the final position, the first end 460a is located on the right side and the other end 460b is located on the left side. This significant change in vessel orientation can facilitate movement of cells across, along, and around the hollow fiber bundle in the culture vessel 200 to facilitate closer proximity of the cells to the nutrient mixture inside the culture vessel 200 (e.g., the extracapillary space 314 as shown in fig. 3), while also facilitating redistribution of the cell population into a less dense manner. In some examples, culture vessel 200 may be further rotated, for example, up to 270 ° or up to 360 °.

Referring again to FIGS. 13A and 13B, in some examples, a sensor 462 (e.g., an optical sensor) can detect the rotational position of the culture vessel 200, which can cause the vessel to stop rotating when rotated to a maximum rotatable position. For example, sensor 462 may detect the position of base 458, e.g., to detect whether a corner of the holder has passed a position in front of sensor 462, thereby indicating the rotational position of culture container 200. In some instances (not shown), rotation of the culture vessel 200 can be stopped mechanically, for example, using an obstruction to prevent rotation beyond a certain degree.

As shown in FIGS. 15A-15C, the exemplary swivel bracket 550 enables the culture vessel 200 to rotate about two separate axes. The swivel bracket 550 includes a housing 552 having a bracket 554 on which the culture vessel 200 may be mounted. A mechanical rotation device (not shown) is mounted within housing 552 and causes culture vessel 200 to rotate about two axes. As shown in FIGS. 15A-15C, the swivel bracket 550 enables the culture container 200 to rotate about an axis passing through the center of the culture container 200, as well as rotate (e.g., twist) about the long axis of the culture container 200. In some examples, the mechanical rotation device of the rotating bracket 550 may rotate about different axes. In some examples, the mechanical rotation device may rotate culture vessel 200 about two axes in a single motion. In some examples, the rotation of culture vessel 200 about one axis may be relatively independent of the rotation of the vessel about another axis. The degree of rotation about each axis may be different. For example, the rotation bracket 550 may be rotated such that the end-to-end rotation angle of the culture container 200 is ± 180 ° and the twist angle is ± 30 °.

In some examples, sensor 562 (e.g., an optical sensor) may detect the rotational position of culture container 200 such that rotation may stop when culture container 200 reaches a maximum rotational angle.

In some examples, the operation of the rotating support (e.g., 450 or 550) can be controlled manually, such as by a handle, lever, or other component that can be operated by a user of the cell culture system. In some examples, the operation of the rotating scaffold may be controlled by a computing device that controls the cell culture system. For example, the rotating bracket may be controlled to rotate the culture vessel by a particular angle at a particular timing schedule. In some examples, rotation may be triggered by a detected cell characteristic parameter in the culture vessel. These characteristic parameters may include cell density in the culture vessel, rate of glucose consumption, rate of lactate accumulation, or an index based on a particular combination of these individual characteristic parameters, or yet other characteristic parameters. For example, an image of the interior of the culture vessel may be captured using an optical sensor (e.g., a still or video camera), analysis of the image may indicate the density of cells in the culture vessel, and rotation may be triggered when the cell density reaches a threshold density.

In some examples, a temperature control system may be used to maintain the temperature of the fresh culture fluid source at a target temperature. For example, by storing the fresh culture fluid source at a low temperature (e.g., a temperature below room temperature), the useful life of the fresh culture fluid source can be extended.

Referring to fig. 16 and 17A-17B, the example temperature control system 650 includes a housing 652 with a source of fresh medium 206 stored in an interior space 654 of the housing. The housing 652 may be, for example, a thermally insulating housing. A liquid amount sensor 656 (e.g., a strain gauge or a volume sensor) may be mounted on the outside or inside of the housing 652 to detect the amount, e.g., weight or volume, of cell culture liquid in the fresh culture liquid source 206.

A temperature control module 658, such as a thermoelectric refrigeration module, such as a Peltier refrigeration module, uses the heat output to control the temperature of the housing interior 654. The output may be a cooling output or a heating output. For example, warm air from the cell culture system (e.g., air warmed by waste heat dissipated by cell culture system components) can be directed to temperature control module 658 by fan 660, and temperature control module 658 can generate a thermoelectric cooling output. The cooling output of temperature control module 658 cools the air stream as air from interior space 654 flows through heat exchanger 670 via fan 672. The cooled air flow is then returned to the interior space 654 and the fresh culture fluid source 206 can be maintained at the target temperature. Similarly, heating may be achieved if the thermoelectric cooling modules are utilized in reverse.

In some examples, the cell culture fluid from the fresh culture fluid source 206 may be heated prior to its introduction into the fluid circuit of the cell culture system. A warming flow path 674 is provided at the output of the fresh broth source 206. The warming flow path 674 may pass through a warming region 676 that may be heated by waste heat from the temperature control module 658 (as shown) or by a separate heating element. In some examples, the heated flow path 674 can include a length of tubing, such as a coil or serpentine tubing, such that the media in the tubing remains in the heated region 676 for a sufficient time to reach a target temperature. In some examples, the heating flow path 674 can include a liquid receptacle (e.g., a thin-walled tank) to facilitate heating of the cell culture fluid. In some examples, the heating of the heating region 676 can be controlled such that the heating region 676 is heated only when cell culture fluid is pumped into the fluid circuit of the cell culture system, e.g., when pumping is not occurring, the cell culture fluid residing in the warming flow path 674 will not be heated.

The operation of temperature control system 650 may be controlled by temperature controller 678. In some examples, as shown in FIG. 16, temperature controller 678 is incorporated into temperature control system 650, which can operate independently of automated cell culture system 100. In some examples, the computing device controlling the operation of the automated cell culture system 100 may also control the operation of the temperature control system 650.

In some examples, a pump (e.g., pump 208a of fig. 2) for pumping fresh cell culture fluid from the fresh culture fluid source 206 into the fluidic circuit may be disposed in the interior space 654 of the temperature control system 650.

In some examples, housing 652 of temperature control system 650 may be divided into a plurality of different interior spaces, which may allow for storage of raw materials at different temperatures. For example, the plurality of distinct interior spaces may store cell culture fluid and cell culture reagents, such as growth factors, serum or other reagents, at appropriate temperatures.

In some examples, temperature control module 658 may include a compartment for cooling material (e.g., dry ice), e.g., in addition to or in place of a thermoelectric cooling module. In some examples, the cooling module may be a cooling system disposed outside of the housing 652, such as a main cooling system that supplies cooling capacity (e.g., cryogenic fluid) through cooling lines to the temperature control system 650 for a plurality of automated cell culture systems 100.

FIG. 18 shows an example overview 250 of a user interface view for a graphical user interface. For example, the user interface may be developed on a PARLAY encrypted web server platform and thereby produce a set of web pages that are displayed on the user interface of an automated cell culture device or on the user interface of another computing device. The user interface (e.g., of the automated cell culture apparatus) may be accessed locally by the user on the automated cell culture apparatus or remotely by an authorized user via a remote computing device. Typically, a user may use instructions (e.g., by pressing or clicking on an icon on a touch screen user interface) to instruct the operation of the automated cell culture system.

In the example of fig. 18, an initial login screen 252 provides secure access to the user interface. When cell culture is not performed, a standby screen 254 is displayed on the user interface. The user can access different pages: for example, the preset page 256, the user may preset an operation parameter, such as a threshold, through the preset page 256; running page 258 on which the user can monitor the status of ongoing cell cultures; a tools page 260 providing access to system tools for the user; a maintenance page 262 providing access to maintenance functions to personnel, such as authorized maintenance engineers; the user may also turn off the automated cell culture system via a power page 266 on the user interface. In fig. 18, reference numeral 264 denotes a user manual.

19-22 are examples of screen shots that may be displayed on a user interface.

FIG. 19 is an overview interface showing the operating parameters of an automated cell culture system, such as run duration, temperature, atmospheric carbon dioxide Concentration (CO) ₂ ) Atmospheric oxygen concentration (O) ₂ ) pH (e.g., colorimetric pH and ionic pH), glucose concentration in cell culture fluid (GLC), lactic acid concentration in cell culture fluid (LAC), partial dissolved oxygen pressure in cell culture fluid (DO), and culture fluid flow rate. Other loading manners of the view interface may display other parameters, or fewer parameters than shown in FIG. 19.

Fig. 20A-20C are control interfaces for temperature, carbon dioxide concentration, pH through which a user can set a target value, an upper threshold for triggering an alarm, and a lower threshold for triggering an alarm. Other parameters, such as oxygen concentration, glucose concentration, lactate concentration, and dissolved oxygen, may be controlled through similar interfaces.

FIG. 21 is a culture medium exchange control interface through which a user can view and set parameters for cell culture medium exchange, including the amount of cell culture medium in the reservoir bag, the volume of cell culture medium to be replaced, the capacity of the waste liquid container, and the amount of cell culture medium cumulatively transferred from the reset amount of waste liquid into the waste liquid container.

Referring to fig. 22, the user interface may display parameters of the cell culture process in real time. For example, the user interface may display a plot of temperature, pH, and dissolved oxygen over time, e.g., as the cell culture progresses.

Examples of the invention

The following example demonstrates the ability to culture living cells in an automated cell culture system using a hollow fiber cartridge.

Example 1-cell count, viability and metabolic parameters of cultured cells.

Jurkat (clone E6-1) is a typical immortalized human T cell line widely used in immunological studies. Jurkat cells were cultured in an automated cell culture system using two different sized hollow fiber cartridges: small size (20 mL culture volume) and large size (70 mL culture volume). The number of cells, viability and metabolic parameters of the cultured cells were determined. The basal medium was DMEM/F12 containing L-glutamine. The circulating culture fluid in the fluid circuit and the space inside the hollow fiber tube contains 5% fetal calf serum and antibiotics.

The inoculated amount of Jurkat cells and fetal bovine serum were injected into the space outside the tube of the hollow fiber cartridge. Fetal bovine serum was added to the outside of the capillaries along with the seeded cells. 10mL of fetal bovine serum was used in a small size culture tube; 35mL of fetal bovine serum was used for the large size culture tube. The culture broth containing 5% fetal bovine serum and antibiotics was circulated in the fluid circuit and the space in the tube of the hollow fiber cartridge at 100 mL/min for 7 days and then increased to 200 mL/min. Fetal bovine serum was supplemented to the outside of the capillary every three days. Glucose and lactate levels were monitored and calculated as mg/day. Cell number and viability were determined using acridine orange/peroxide iodide staining with a Countless FL II automated cell counter.

FIG. 23A shows total cell count 10 and viable cell count 12 in small size culture tubes; fig. 23B shows total cell count 14 and viable cell count 16 in large format culture tubes. As shown in fig. 23A and 23B, the cell count steadily increased and almost all cultured cells survived.

FIGS. 24A and 24B show the metabolic characteristics of cultured cells in small-size culture tubes and large-size culture tubes, respectively. FIG. 24A shows that glucose uptake 18 and lactate secretion 20 of cells in small size culture tubes continued to increase until day 12 of culture, after which glucose uptake and lactate secretion began to decrease. FIG. 24B shows that glucose uptake 22 and lactate secretion 24 by cells in large culture tubes continued to increase until day 13 of culture, after which glucose uptake and lactate secretion became substantially stable.

Example 2 Effect of dissolved oxygen concentration on T lymphocyte culture

Primary human T lymphocytes were cultured in an automated cell culture system using a hollow fiber cartridge to study the effect of dissolved oxygen concentration on cell expansion. Human T lymphocytes from healthy donors were seeded in the space outside the tubes of the hollow fiber cartridge. In some culture tubes, cells were cultured in AIM-V medium and human AB serum; in some culture tubes, the cells are in the absenceCulturing in the presence of serum X-Vivo 15 culture medium. Interleukin-2 (IL-2) was injected into the tube outer space of the hollow fiber tube every day. For each cell culture fluid, the dissolved oxygen level was controlled at atmospheric environmental level (. About.20%) ₂ ) Or physiological level (5%O) ₂ ). The number of cells in each culture tube was estimated daily.

FIG. 25A shows T cells cultured in AIM-V broth and human AB serum at hypoxia 30 (. About. 5%O) ₂ ) And normoxia 32 (-20%) ₂ ) Cell count under conditions. FIG. 25B shows T cells cultured in serum-free X-Vivo 15 medium at hypoxia 34 (. About. 5%O) ₂ ) And normoxic 36 (-20%) O ₂ ) Cell count under conditions. For both cell culture media, the cell count of T cells cultured under hypoxic conditions was higher than that under normoxic conditions, although the cell count of T cells cultured in AIM-V medium and human AB serum was higher than that cultured in serum-free medium. FIG. 25C shows the fold expansion of cells cultured under hypoxic and normoxic conditions in two cell culture media. Consistent with fig. 25A and 25B, the fold expansion of cells cultured under hypoxic conditions was higher in both cell culture media than under normoxic conditions, and the fold expansion of cells cultured in serum-containing media was also higher than that of cells cultured in serum-free media. These results indicate that physiological oxygen levels greatly increase the cell growth rate and ultimate yield of human T lymphocytes in both serum-containing and serum-free media.

Example 3 expansion of T lymphocytes in an automated cell culture System

Human T lymphocytes were cultured in an automated cell culture system using a hollow fiber cartridge to verify the performance of the automated cell culture system and the hollow fiber cartridge.

In the first validation, human T lymphocytes were cultured in AIM-V medium containing human AB serum. Human T lymphocytes from three healthy donors were treated with Dynabeads CD3/CD28 at 1:1, activated and cultured for 3 days at a ratio of 2X 10 ⁷ The concentration of individual cells/culture tubes is seeded in the capillary outer space of the hollow fiber cartridge. The basic AIM-V culture solution passes through the hollow fiber tubeThe tube inner space of the cartridge circulates in the fluid circuit. Every three days 10mL of human AB serum was injected into the tube outer space of the culture tube. IL-2 was injected into the extratubal space daily in 1.5-fold increments. Cell number and viability were measured using a Countess FL II cytometer. Subpopulations of T lymphocytes were stained with fluorochrome-labeled antibodies targeting CD3, CD4 and CD9 and analyzed with a BD Accuri C6 flow cytometer.

Fig. 26A shows the increase in cell count over time for T cell cultures from three healthy donors. Fig. 26B shows the fold expansion and doubling time of T cell cultures from three donors. Fig. 26C shows cell viability of T cells from three donors at the time of seeding and harvesting. Figure 26D shows the percentage of CD3+ in Peripheral Blood Mononuclear Cells (PBMCs), seeded cells, and harvested cells from three donors. FIG. 26E shows the subset percentages of CD4+ and CD 4-in the CD3+ subpopulations of seeded and harvested cells from PMBC from three donors. These results demonstrate that cells can be expanded and remain viable in an automated cell culture system.

In the next validation, human T lymphocytes were cultured in serum-free X-Vivo 15 medium. Human T lymphocytes from three healthy donors were treated with Dynabeads CD3/CD28 at a ratio of 1:1, activated and cultured for 3 days at a ratio of 2X 10 ⁷ The concentration of individual cells/culture tubes is seeded in the outer space of the hollow fibre cartridge. The basic X-Vivo 15 culture fluid circulates in the fluid circuit via the outer space of the hollow fiber cartridge. Every three days, 10mL of 1% human albumin solution was injected outside the capillaries of the culture tubes. IL-2 was injected daily into the outside of the capillaries in 1.5-fold increments. Cell number and viability were measured using a Countess FL II cytometer. Subpopulations of T lymphocytes were stained with fluorochrome-labeled antibodies targeting CD3, CD4 and CD9 and analyzed with a BD Accuri C6 flow cytometer.

Fig. 27A shows the increase in cell count over time in T cell cultures from three healthy donors. Figure 27B shows fold expansion and doubling time of T cells from three donors. Fig. 27C shows T cell viability of T cells from three donors at the time of seeding and harvesting. Figure 27D shows the CD3+ percentage of PBMCs, seeded cells and harvested cells from three donors. FIG. 27E shows the subset percentages of CD4+ and CD 4-in the CD3+ subpopulations of seeded and harvested cells from PMBC from three donors. These results demonstrate that cells can be expanded and can retain viability in an automated cell culture system.

A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. For example, some of the steps described above are ordered chronologically, and thus can be performed in a different order than illustrated in the description.

Other implementations are within the scope of the following claims.

Claims

1. An automated cell culture system for culturing T cells, comprising:

a cell culture reactor comprising:

a housing;

a fluidic circuit for a cell culture fluid, the fluidic circuit being disposed inside the housing and comprising:

a culture vessel for culturing cells in a cell culture solution,

a receptacle for cell culture fluid, the receptacle being in fluid communication with the culture vessel, and

a pump for pumping cell culture fluid into the fluid circuit;

one or more sensors disposed inside the housing and coupled to a computing device of the automated cell culture system, each sensor for detecting in real time one or more of (1) a cell culture fluid in the fluid circuit and (2) a parameter in an environment inside the housing;

the computing device receiving signals generated by one or more detected parameters from the one or more sensors to automatically control operation of the cell culture reactor, wherein the computing device is configured to: determining a stage of cell culture in the culture vessel based on one or more of (i) one or more detected parameters and (ii) a history of the one or more detected parameters; and controlling operation of the cell culture reactor based on the stage of cell culture, the one or more detected parameters including broth volume, fluid pressure, flow rate, pH, dissolved oxygen, glucose concentration, lactate concentration;

a gas source in fluid communication with the interior of the housing; and

a gas flow control device connected to a gas source;

wherein the computing device is configured to control operation of the airflow control device based on the one or more detected parameters,

wherein the computing device receives a signal from the carbon dioxide sensor and controls operation of the carbon dioxide mass flow controller in response to the signal to maintain the concentration of carbon dioxide within a predetermined range, and

wherein the computing device receives a signal from the oxygen sensor and controls operation of the nitrogen mass flow controller in response thereto to maintain the concentration of oxygen within a predetermined range,

wherein the dissolved oxygen level is controlled at physiological level 5%O ₂ ，

Wherein the computing device controls operation of the cell culture reactor based on a comparison between one or more of the detected parameters and respective thresholds.

2. The automated cell culture system for culturing T cells of claim 1, comprising a rotating rack for the cell culture vessel.

3. An automated cell culture system for culturing T cells according to claim 1 comprising a feed system comprising:

a supply line having one end connected to the fluid circuit and the other end connected to a cell culture fluid source;

a supply pump connected to the supply line; and

a temperature control system, comprising:

a housing, an inner space of the housing for accommodating a cell culture fluid source; and

and a temperature control module for cooling or heating the inner space of the housing.

4. The automated cell culture system for culturing T cells of claim 3, wherein the computing device controls operation of the feed pump as a function of one or more of (i) the amount of cell culture fluid in the receptacle and (ii) the pH of the cell culture fluid in the fluidic circuit.

5. The automated cell culture system for culturing T cells of claim 1, comprising a heater disposed inside the housing.

6. The automated cell culture system for culturing T cells of claim 1, comprising a valve in the housing, wherein the computing device is configured to control operation of the valve based on a concentration of a gas in an interior of the housing.

7. The automated cell culture system for culturing T cells of claim 1, wherein the culture vessel comprises a hollow fiber cartridge.

8. A method of culturing T cells, the method comprising:

incubating T cells in a cell culture reactor comprising:

flowing a cell culture fluid in a fluid circuit inside a cell culture reactor, comprising pumping the cell culture fluid from a reservoir of cell culture fluid into a culture vessel to culture T cells in the cell culture fluid;

detecting in real-time, by each of one or more sensors disposed inside the cell culture reactor and coupled to a computing device, one or more parameters of (1) a cell culture fluid in a fluidic circuit and/or (2) an environment inside the cell culture reactor; and

automatically controlling, by the computing device, operation of the cell culture reactor by receiving signals generated by one or more detected parameters from the one or more sensors;

wherein the computing device is configured to: determining a stage of cell culture in the culture vessel based on one or more of (i) one or more detected parameters and (ii) a history of the one or more detected parameters; and controlling operation of the cell culture reactor based on the stage of cell culture, the one or more detected parameters including broth volume, fluid pressure, flow rate, pH, dissolved oxygen, glucose concentration, lactate concentration,

wherein controlling operation of the cell culture reactor comprises controlling operation of one or more gas flow control devices and valves connected to a gas source and the cell culture reactor housing,

Wherein controlling operation of the cell culture reactor comprises: comparing each detected parameter to a respective threshold value; controlling operation of the cell culture reactor based on the comparison.

9. The method of claim 8, comprising rotation of the culture vessel.

10. The method of claim 8, wherein the controlling operation of the cell culture reactor comprises controlling operation of a feed pump to pump cell culture fluid from a cell culture fluid source into the fluidic circuit in accordance with a control algorithm comprising one or more of (i) an amount of cell culture fluid in the receptacle and (ii) a pH of the cell culture fluid in the fluidic circuit.

11. The method of claim 10, comprising controlling the temperature of the cell broth source.

12. The method of claim 8, wherein controlling operation of the cell culture reactor comprises controlling a heater based on a temperature in the cell culture reactor.

13. The method of claim 8, comprising outputting information or an alert based on the detected one or more parameters.

14. The method of claim 8, comprising receiving input through a user interface or from a remote computing device; and further controlling operation of the cell culture reactor based on the received input.